Field of the Invention
The disclosure provides a method for generation of humanized full length antibodies in mammalian cells. A library of humanized variants is provided with high, validated framework diversity without requiring back-mutations to retain original affinity. Synthetic CDR encoding fragment libraries derived from a template antibody are ligated to human framework region encoding fragments from a human framework pool limited only to germline sequences from a functionally expressed antibodies. The vector comprises a nucleic acid sequence encoding HC framework region 4. No CDR grafting or phage display is required.
Description of the Related Art
Monoclonal antibodies (MAbs) are monospecific for a particular antigen and are made by identical immune cells that are clones of a unique parent cell. Monoclonal antibodies traditionally are made by fusing myeloma cells with spleen cells from a mouse that has been immunized with the desired antigen. Fused hybrid cells, or hybridomas, can be grown indefinitely in cell culture media, or can be injected in mice where they produce tumors containing an antibody rich fluid called ascites fluid. Antibodies can then be purified from the cell culture medium or ascites.
Monoclonal antibody therapy is the use of MAbs to specifically bind to an antigen, for example, a cell surface antigen on a target cell. This may stimulate the patient's immune system to attack those cells. MAbs have been developed to treat various diseases such as rheumatoid arthritis, multiple sclerosis and different types of cancers. Initially, murine antibodies were obtained with hybridoma technology; however the dissimilarity between murine and human immune systems resulted in several clinical failure of these antibodies. Nevertheless, a small number of murine MAbs are FDA approved to treat various conditions. Muromonab-CD3 (Orthoclone OKT3) is a murine MAb that targets the T cell CD3 receptor and was approved in 1986 for transplant rejection. Tositumoman (Bexxar) is a murine Mab that targets CD20 and was approved in 2003 for the treatment of Non-Hodgkin lymphoma.
Therapeutic deficiencies of mouse monoclonal antibodies as human therapeutics are well known and include short in vivo half life, weak effector functions mediated by the mouse heavy chain constant region; patient sensitization to the antibody, and generation of a human anti-mouse antibody (HAMA) response; and neutralization of the mouse antibody by HAMA leading to a loss of therapeutic efficiency. See, for example, Williams et al. 2010, Humanising antibodies by CDR grafting. Antibody Engineering, Edit by R. Kontermann and S. Dubel, Springer Lab Manual, 319-339. One major obstacle in early development of therapeutic antibodies was the human anti-murine antibody (HAMA) response which occurred in up to about 50% of patients upon administration of murine hybridoma-derived antibodies and compromised the safety, efficacy and half-life of the antibody therapeutics.
One way to alleviate certain deficiencies of mouse monoclonal antibodies is antibody humanization. Various techniques of antibody humanization are known. One method of antibody humanization is chimerization. In mouse/human chimeric antibodies, the immunogenic murine constant domains are replaced by the human counterpart. Intact murine variable domains are preserved to maintain the intrinsic antigen-binding affinity; i.e., the entire Fv regions were retained from the murine antibody (about 66% human). Antibody chimerization was found to alleviate the short in vivo half-life compared to the murine MAb, and impart human Fc effector function on the antibody. Although chimerization of some murine antibodies resulted in reduced HAMA response, others remained immunogenic. A few chimeric antibodies are FDA approved to treat various conditions. Abciximab (ReoPro), is a chimeric antibody which targets inhibition of glycoprotein IIb/IIIa and was FDA approved in 1994 for the treatment of cardiovascular disease. Infliximab (Remicade) is a chimeric antibody that results in inhibition of TNF-alpha signaling; was first approved in 1998 and is now used for the treatment of several autoimmune disorders.
A second technique of antibody humanization termed CDR grafting involves the transplantation of the entire murine CDRs onto a human framework region wherein the reshaped humanized antibody only retained essential binding elements from the murine antibody (5-10% of the total sequence). For example, see Lo, Antibody humanization by CDR grafting. Antibody Engineering, Methods and protocols. Edit by Benny K. C. Lo, Methods in Molecular Biology, 2004, 248, 135-159. CDR grafting is described in U.S. Pat. Nos. 5,225,539 and 5,585,089, each of which is incorporated herein by reference. Humanized antibodies from CDR grafting resulted in increased in vivo tolerance and efficacy of therapeutic antibodies. According to Lo 2004, the key to successful CDR grafting lies in the preservation of the murine CDR conformations in the reshaped antibody for antigen binding. The antibody Fv region comprises variable domains from the light chain (VL) and the heavy chain (VH) and confers antibodies with antigen-binding specificity and affinity. The variable domains adopt the immunoglobulin fold in which two antiparallel beta-sheet framework scaffolds support three hypervariable CDRs. Unfortunately, CDR grafting often leads to suboptimal orientations of the murine CDR loops responsible for antigen binding. Therefore, critical murine framework residues needed to be reintroduced as back-mutations to restore the optimum CDR conformations for antigen binding. According to Williams et al. 2010, ibid., at least 118 antibodies have been humanized. Of the 24 approved antibodies on the market, 13 are humanized, four are murine, five are chimeric and two are human. The marketed, humanized antibodies were all generated by CDR grafting.
Another technique for developing a minimally immunogenic humanized antibody is known as SDR grafting. Some humanized antibodies were found to elicit an anti-idiotype (anti-Id) response against the potentially immunogenic murine CDRs. Further, it was found that not all of the CDRs are equally important, or even essential, for antigen binding. It was also found that only about 20-33% of CDR residues are involved in antigen contact. The CDR residues that are most important in antigen-antibody interaction are called specificity-determining residues (SDRs). SDRs are found at positions of high variability and may be determined by determination of the three-dimensional structure of the antigen-antibody complex or by genetic manipulation of the antibody-combining site. See for example, Kashmiri et al., 2004, Developing a minimally immunogenic humanized antibody by SDR grafting. Antibody Engineering, Methods and protocols. Edit by Benny K. C. Lo, Methods in Molecular Biology, 248, 361-376. Therefore, there is room for protein evolution within the CDR regions while maintaining affinity for the target antigen.
Competing technologies exist to reduce the immunogenicity of antibodies. Transgenic mice (e.g. Xenomouse and UltiMAb-Mouse) containing large parts of the human immunoglobulin locus, can be a source of “fully human” antibodies. Human antibodies derived from a bacteriophage library of human variable regions also need no humanization, but frequently need further mutation to achieve high binding potency. A relatively small number of marketed antibodies have been derived using these other platform technologies. See Williams et al. 2010, ibid.
According to Lo 2004, the goal of antibody humanization is to engineer a monoclonal antibody (MAb) raised in a non-human species into one that is less immunogenic when administered to humans. However, it would be advantageous to develop a technique of antibody humanization that is accompanied by various protein evolutionary techniques to produce a humanized antibody with other optimized characteristics such as enhanced affinity for the target antigen compared to the mouse and/or increased expression.